Billed as "A Musical Play (Fantasy) in Two Acts, Nine Scenes," Cabin in the Sky opened on 25 October 1940 at New York's Martin Beck Theater. The playbill on opening night included the following credits: "Book by Lynn Root. (Based on the story "Little Joe" by Lynn Root.) Music by Vernon Duke. Lyrics by John Latouche. Entire production staged by George Balanchine. Settings and costumes by Boris Aronson. Produced by Albert Lewis in association with Vinton Freedley." The undisputed star of the show was Ethel Waters, whose name appeared above the title. Other principal players were Dooley Wilson, Rex Ingram, Katherine Dunham, J. Rosamund Johnson, and Todd Duncan. Besides supporting players, the all-black cast included the Katherine Dunham Dancers, and the J. Rosamund Johnson Singers.

What the published credits do not reveal is that George Balanchine and Katherine Dunham were, in effect, co-choreographers of the dances in the show, at least for those in which she and her dancers appeared. When choreographing for dancers trained in techniques other than classical ballet, Balanchine's habit was to respect their expertise and their personal style, to allow them as much creative input as they wished to make, and then to arrange their steps, combinations, and movements into a unified choreographic composition. Dunham found this method of collaboration quite agreeable, and she and Balanchine enjoyed a particularly amicable working relationship.

The story of Cabin in the Sky centers on Little Joe, a kindhearted but morally ambivalent Everyman, who is stabbed in a dispute over a crap game, dies and is bound for Hell, but is saved by his good wife's prayers and given extra time on earth to qualify for admission to Heaven. Dooley Wilson played Little Joe; Ethel Waters was his faithful wife Petunia; and Katherine Dunham was the sexy temptress Georgia Brown, assistant to Lucifer Jr., played by Rex Ingram. Todd Duncan portrayed The Lawd's General.

The Dunham Dancers were featured in "Dem Bones" and "Do What You Wanna Do" in act 1 and in three production numbers in act 2: the Egyptian Ballet, Lazy Steps, and Boogy Woogy. In act 2 Dunham and Dooley Wilson sang "Love Me Tomorrow," and Dunham, supported by some of her male dancers, performed a saucy song-and-dance number to "Honey in the Honeycomb." (She would later perform this number as a solo in nightclubs.) The big hit song of the show, however, was "Taking a Chance on Love," sung by Ethel Waters, who also sang "Cabin in the Sky," "Holy unto the Lord," "Dem Bones," "My Old Virginia Home on the Nile," "Love Turned the Light Out," and "Savannah," during which she danced a honky-tonk version of the samba.

When the show ended its Broadway run on 8 March 1941, after 156 performances, the company set out on a national tour.